Volume 100, Issue 5, 2 March 2011, Pages 1252–1260
Bilayer-Mediated Clustering and Functional Interaction of MscL Channels
Abstract
Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.
Introduction
The mechanosensitive channel of large conductance (MscL) is a homopentameric protein that is gated by membrane tension. It primarily acts as an emergency relief valve to protect bacterial cells from lysis during periods of hypoosmotic shock (1). In its open conformation, the channel has a nonselective pore ∼3 nm in diameter (2 and 3) that allows the rapid escape of cytoplasmic osmolytes.
Clustering of channels has been demonstrated in several systems, most notably acetylcholine receptors and glycine receptors, and in both cases the underlying mechanism was attributed to specialized proteins, such as gephyrin (4 and 5), that specifically anchor the receptors at predetermined locations. Lipid raft formation has been postulated as another mechanism that leads to membrane-protein clustering (e.g., for the sodium-potassium ATPase in cerebellar granular cells (6)). More recently, very weak homophilic protein-protein interactions were shown to lead to significant protein self-assembly within membranes, as observed for syntaxins (7). Clustering of membrane proteins due to membrane-mediated interactions has also been observed in ryanodine receptors (8 and 9), gramicidin A channels (10), the bacterial potassium channel KcsA (11), and rhodopsin (12). Although there is increasing evidence that proteins in general assemble in clusters (13), clustering has not yet been demonstrated for the majority of membrane proteins, including MscL.
As is the case for most membrane proteins, the function of MscL has been regarded as a result of its architecture and response to the lipid environment (14 and 15). Most studies have focused on individual proteins and assumed no interactions with neighboring proteins. In the study presented here, we systematically obtained observations that are not in accordance with results obtained from isolated channels. Instead of independent and randomly distributed channels, we observed the formation of MscL clusters in all of the lipid systems examined, using a combination of structural techniques (small-angle neutron scattering (SANS), atomic force microscopy (AFM), and neutron reflection), fluorescence microscopy, and patch-clamp recording.